The present invention relates to compositions and methods for treatment of occlusive peripheral vascular disease and coronary diseases, in particular, the occlusion of coronary vessels. More particularly, the invention relates to the promotion of the growth of new blood vessels (angiogenesis), especially coronary blood vessels, and/or the recruitment of collateral blood vessels, after myocardial infarction.
It is estimated that five million people are afflicted with chronic stable angina in the United States. Each year 200,000 people under the age of 65 die with what is termed xe2x80x9cpremature ischemic heart disease.xe2x80x9d Despite medical therapy, many go on to suffer myocardial infarction and debilitating symptoms prompting the need for revascularization with either percutaneous transluminal coronary angioplasty or coronary artery bypass surgery. Medical researchers have postulated that one way of relieving myocardial ischemia would be to enhance coronary collateral circulation.
Fujita et. al. (Fujita et al., Am. Heart Journal., 122:453 (1991), Fujita et al., Int. J. Cardiol., 40:51 (1993)) demonstrated that heparin in combination with short term exercise training improved exercise tolerance as measured by dynamic exercise testing. The researchers, believing this effect was mediated through increased collateral vascular development, examined the effects of heparin in combination with a brief concomitant exercise training protocol on coronary collateral flow. Thallium-201 myocardial perfusion images obtained in association with the same work-load both before and late after combined heparin exercise treatment, which indicated that coronary collateral circulation was enhanced. Such dramatic changes over a short term do not occur naturally, and suggest that angiogenesis has taken place. These investigators carried out further studies which demonstrated that exercise alone or heparin alone were insufficient stimuli for collateral development (Fujita et al., Am. Heart Journal, 122:453 (1991)). That is, only when exercise and heparin were combined were they able to elicit this apparent angiogenic response. Other studies have suggested that exercise-induced ischemia combined with heparin increases coronary collateral flow.
More recently Quyyumi et. al. (Quyyumi et al., J. Am. Coll. Cardiol., 22:635 (1993)) studied the anti-ischemic effects of combined treatment with low molecular weight heparin and exercise-induced ischemia. Twenty three patients received either heparin or placebo in combination with an exercise protocol for 4 weeks. Eighty percent of the low molecular weight heparin (LMWH) group compared with 31% of placebo group had a significant increase in rate-pressure product at the onset of 1 mm of ST segment depression. Further, the time to ischemia increased in 100% of the LMWH group compared with 62% in the placebo group. In this same population, the incidence and duration of ST segment depression, measured using an ambulatory holter monitor, decreased by 30 and 35% respectively compared with 0% in controls.
These authors concluded that exercise and LMWH lessens myocardial ischemia and that the improvement is likely to be mediated by enhanced collateral function. Similar findings resulted from another double-blind, randomized, placebo-controlled trial, involving 29 patients with stable exercise-induced angina pectoris who received a single daily subcutaneous injection of LMWH Pamaparin (trademark for a brand of heparin)
Correlations have now been made between the anatomic appearance of coronary collateral vessels (xe2x80x9ccollateralsxe2x80x9d) visualized at the time of intracoronary thrombolitic therapy during the acute phase of myocardial infarction and the creatine kinase time-activity curve, infarct size, and aneurysm formation. These studies demonstrate a protective role of collaterals in hearts with coronary obstructive disease, showing smaller infarcts, less aneurysm formation, and improved ventricular function compared with patients in whom collaterals were not visualized.
When the cardiac myocyte is rendered ischemic, collaterals develop actively by growth with DNA replication and mitosis of endothelial and smooth muscle cells. One hypothesis suggests that heparin-binding growth factors are present in the heart, or that biological activity is quiescent under normal physiological conditions. Once ischemia develops, these factors are activated and become available for receptor occupation, which may initiate angiogenesis after exposure to exogenous heparin. Unfortunately, the xe2x80x9cnaturalxe2x80x9d process by which angiogenesis occurs is inadequate to reverse the ischemia in almost all patients with coronary artery disease.
The etiology of the benefit of combined heparin-exercise treatment is unknown with certainty (Norrby and Sorbo, Int. J. Exp. Pathol. 73: 147 (1992), Sasayama and Fujita, Circulation, 85: 1197 (1992)). One possibility is that ischemia stimulates the release or expression of some angiogenic substance which in combination with heparin stimulates collateral development. However, a definitive link between an angiogenic substance and heparin to promote angiogenesis has not been established.
During ischemia, adenosine is released through the breakdown of ATP. Adenosine participates in many cardio-protective biological events. Adenosine has a role in hemodynamic changes such as bradycardia and vasodilation, and adenosine has been suggested to have a role in such unrelated phenomena as preconditioning and possibly the reduction in reperfusion injury (Ely and Beme, Circulation, 85: 893 (1992)).
Intrinsic adenosine may facilitate the coronary flow response to increased myocardial oxygen demands and so modulate the coronary flow reserve. Ethier et. al. (Ethier et al., Am. J. Physiol., H131 (1993)) demonstrated that the addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor. They suggested that adenosine may be a factor for human endothelial cell growth and possibly angiogenesis. Angiogenesis appears to be protective for patients with CAD, but the rate at which blood vessels grow naturally is inadequate to reverse the disease. Thus, strategies to enhance and accelerate the body""s natural angiogenesis potential should be beneficial in patients with CAD.
Combinations of thrombolytic agents such as streptokinase, urokinase and tissue plasminogen activator with adenosine have been proposed for use in providing coronary thrombolysis (see, for example, U.S. Pat. No. 5,534,504 to Sollevi). Sollevi does not teach that these agents, in combination with adenosine, provided any angiogenic benefit. Sollevi further teaches that administration of heparin is unsafe, and instead teaches administering adenosine in lieu of heparin.
There remains a need for an effective therapy for promotion of coronary angiogenesis with minimum side effects. Such a therapy would be particularly useful for patients who have myocardial infarctions and could be used prophylactically in patients who have poor coronary circulation which places them at high risk of ischemia and myocardial infarctions.
Compositions and methods for treatment of occlusive peripheral vascular disease and coronary diseases, in particular, the occlusion of coronary vessels, and disorders associated with the occlusion of the peripheral vasculature and/or coronary blood vessels, are disclosed. Also disclosed are compositions and methods for promoting angiogenesis and/or recruiting collateral blood vessels in a patient in need thereof. The compositions include an effective amount of heparin or a heparin-like substance and an effective amount of an adenosine A2 receptor agonist. The compositions can be in the form of a sterile, injectable, pharmaceutical formulation that includes an angiogenically effective amount of heparin or a heparin-like substance and an adenosine A2 receptor agonist in a physiologically and pharmaceutically acceptable carrier, optionally with one or more excipients.
The methods involve the co-administration of an effective amount of heparin or a heparin-like substance and an effective amount of an adenosine A2 receptor agonist in low, daily dosages for a week or more. One or both components can be delivered locally via catheter. Heparin (or heparin-like substances) and relatively stable adenosine A2 agonists (i.e., those with a half-life greater than about 15 minutes in vivo can be delivered to capillary beds surrounding ischemic tissue by incorporation of the compounds in an appropriately sized liposome or microparticle. Heparin can be targeted to ischemic tissue by covalent linkage with a suitable antibody.
The method may be used as a treatment to restore cardiac function after a myocardial infarction. The method may also be used to improve blood flow in patients with coronary artery disease suffering from myocardial ischemia or inadequate blood flow to areas other than the heart, for example, occlusive peripheral vascular disease (also known as peripheral arterial occlusive disease), where decreased blood flow is a problem.
Compositions and methods for treatment of occlusive peripheral vascular disease and coronary diseases, in particular, the occlusion of coronary vessels, and disorders associated with the occlusion of the peripheral vasculature and/or coronary blood vessels, are disclosed. Also disclosed are compositions and methods for promoting angiogenesis and/or recruiting collateral blood vessels in a patient in need thereof. The compositions include an effective amount of heparin or a heparin-like substance and an effective amount of an adenosine A2 receptor agonist. The methods involve the co-administration of an effective amount of heparin or a heparin-like substance and an effective amount of an adenosine A2 receptor agonist in low, daily dosages for a week or more.
Definitions
As used herein, the term xe2x80x9cmyocardial ischemiaxe2x80x9d is defined as an insufficient blood supply to the heart muscle caused by a decreased capacity of the heart vessels.
As used herein, the term xe2x80x9ccoronary diseasexe2x80x9d is defined as diseases/disorders of cardiac function due to an imbalance between myocardial function and the capacity of coronary vessels to supply sufficient blood flow for normal function. Specific coronary diseases/disorders associated with coronary disease which can be treated with the compositions and methods described herein include myocardial ischemia, angina pectoris, coronary aneurysm, coronary thrombosis, coronary vasospasm, coronary artery disease, coronary heart disease, coronary occlusion and coronary stenosis.
As used herein the term xe2x80x9cocclusive peripheral vascular diseasexe2x80x9d (also known as peripheral arterial occlusive disorder) is a vascular disorder involving blockage in the carotid or femoral arteries, including the iliac artery. Blockage in the femoral arteries causes pain and restricted movement. A specific disorder associated with occlusive peripheral vascular disease is diabetic foot, which affects diabetic patients, often resulting in amputation of the foot.
As used herein the terms xe2x80x9cregeneration of blood vessels,xe2x80x9d angiogenesis,xe2x80x9d xe2x80x9crevascularization,xe2x80x9d and xe2x80x9cincreased collateral circulationxe2x80x9d (or words to that effect) are considered as synonymous. The term xe2x80x9cpharmaceutically acceptablexe2x80x9d when referring to a natural or synthetic substance means that the substance has an acceptable toxic effect in view of its much greater beneficial effect, while the related the term, xe2x80x9cphysiologically acceptable,xe2x80x9d means the substance has relatively low toxicity. The term, xe2x80x9cco-administeredxe2x80x9d means two or more drugs are given to a patient at approximately the same time or in close sequence so that their effects run approximately concurrently or substantially overlap. This term includes sequential as well as simultaneous drug administration.
As used herein, the term xe2x80x9cheparin-like substancexe2x80x9d refers to compounds which mimic the action of heparin. These include heparin-like glycosaminoglycans such as chondroitin sulfates; dermatan sulfates; heparan sulfates; low molecular mass heparin fragments such as ardeparin sodium, de-N-sulfated heparin, nitrous-acid deaminated heparin, and periodate-oxidized heparin; heparin fractions, and heparin salts such as ammonium, calcium, lithium, sodium, and zinc. The heparin-like substances preferably provide an anti-Xa Activity and anti-IIa activity similar to that of heparin.
Other conventional anti-coagulants such as hirudin, ancrod, warfarin, tissue plasminogen factor, streptokinase, urokinase and Integrilin(trademark) (commercially available from Cor Therapeutics), and combinations thereof are not intended to be equivalents of heparin, because they may exert their anti-coagulative effects by an entirely different mechanism. However, these can be present as optional components.
As used herein, a compound is an agonist of an adenosine A2 receptor if it is able to fully inhibit adenylate cyclase and is able to displace [125I]-AB-MECA in a competitive binding assay. The agonist can be effective toward the A2a or the A2b receptor.
A selective A2 receptor agonist is one which has a ratio of A2/A1 activity greater than 50 and a ratio of A2/A3 activity greater than 50.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d refers to pharmaceutically acceptable salts of heparin, a heparin-like substance, or an adenosine A2 receptor agonist, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can be used as the pharmaceutically acceptable salt.
A. Heparin
Heparin is a heterogeneous mixture of polysaccharides derived from beef or pork livers. Although the exact mechanism for heparin""s antithrombotic properties is not known, it is believed to act by binding to antithrombin III. The heparin-antithrombin III complex inhibits the activity of numerous enzymes in the clotting cascade, including factors IIa (thrombin), IXa, Xa, XIa, and XIIa (Carter et al. xe2x80x9cEnoxaparin: The low-molecular-weight heparin for prevention of postoperative thromboembolic complications,xe2x80x9d Ann. Pharmacother., 27:1223-30 (1993); Olin, ed. Drug Facts and Comparisons. St. Louis: Facts and Comparisons, Inc., 1997:86b-g; Fareed and Hoppensteadt, xe2x80x9cPharmacology of the low-molecular-weight heparins,xe2x80x9d Semin. Thromb. Hemostasis. 22(Suppl 2):13-8 (1996); Fareed et al. xe2x80x9cAre the available low-molecular-weight heparin preparations the same?xe2x80x9d Semin. Thromb. Hemostasis, 22(Suppl 1):77-91 (1996); and Buckley and Sorkin, xe2x80x9cEnoxaparin: A review of its pharmacology and clinical applications in the prevention and treatment of thromboembolic disorders,xe2x80x9d Drugs, 44:465-97 (1992)). In addition, heparin induces release of other endogenous antithrombotic substances, such as tissue factor pathway inhibitor and tissue plasminogen activator.
The effective dose of heparin can vary widely from patient to patient. A small percentage of patients who are administered heparin over an extended period of time develop heparin-induced thrombocytopenia (HIT). For this reason, it may be advantageous, at least for certain patients, to administer heparin-like substances instead of heparin. Suitable heparin-like substances are disclosed in detail below.
B. Chondroitin Sulfates
Chondroitin sulfates are structurally complex, sulfated, linear polysaccharides known as galactosaminoglycans (GAGS) comprising alternating uronic acid and N-acetyl-D-galactosamine residues. Chondroitin sulfates are localized on cell surfaces and in the extracellular matrix, and are important in cell to cell communications. They are the predominant GAGS comprising the proteoglycans produced by monocyte/macrophages.
Chondroitin Sulfate A (CSA) includes unsulfated glucuronic acid 1xe2x86x923 linked to 4-O-sulfated N-acetyl-D-galactosamine which in turn is attached to the next glucuronic acid by a 1xe2x86x924 linkage. Chondroitin Sulfate B, also known as Dermatan Sulfate or beta-heparin, is similar to CSA except that it contains iduronic acid instead of glucuronic acid. Chondroitin Sulfate C (CSC) has a 6-O-sulfate group and Chondroitin Sulfate E has a 4,6-di-O-sulfate on N-acetyl-D-galactosamine, in place of a 4-O-sulfate found in CSA. Suitable chondroitin sulfates include those described in Bjornsson et al., xe2x80x9cThe Anticoagulant Effect of Chondroitin-4-Sulfate, Thromb Res., 27: 15-21 (1982); U.S. Pat. No. 3,895,106 to Morrison, Mazieres et al., xe2x80x9cChondroitin sulfate in the treatment of gonarthrosis and coxarthrosis,xe2x80x9d Rev. Rhum. Mal. Osteoartic., 59: 466-72 (1992); and Nadkarni et al., xe2x80x9cPreparation and biological activity of N-sulfonated chondroitin and dermatan sulfate derivatives,xe2x80x9d Carbohvdrate Res., 290:87-96 (1996), the contents of which are hereby incorporated by reference.
C. Dermatan Sulfates
Dermatan sulfate, also known as xcex2xcex2-heparin or chondroitin sulfate B, is a polysaccharide composed of repeating uronic acid xe2x86x92N-acetyl-D-galactosamine disaccharides joined by 1,3 and 1,4 linkages. It is initially formed as a polymer composed of repeating glucuronosylxe2x86x92galactosylxe2x86x92galactosylxe2x86x92xylosyl linkage regions. In its biosynthesis, some of the D-glucuronic acid residues are epimerized at C-5, converting them to L-iduronic acid residues, which is then followed by O-sulfation primarily at C-4, but also at C-6. Dermatan sulfate functions as an anticoagulant by catalyzing the inhibition of thrombin as it is formed in plasma. It specifically activates heparin cofactor II (HCII), a plasma protease inhibitor which inhibits thrombin but not other proteases involved in hemostasis. HCII is activated by fractions of 12 or more residues in length that contain an octasaccharide sequence required for binding to the inhibitor.
Suitable dermatan sulfates include those disclosed in Tollefsen, xe2x80x9cHeparin and Related Polysaccharides,xe2x80x9d Lane DA, Bjxc3x6xc3x6rk 1, Lindahl U (Eds), Plenum Press, N.Y., pp 167-76 (1992), Van Gorp, xe2x80x9cHeparins and Structurally-Related Glycosaminoglycans,xe2x80x9d Clin. Hemost. Rev. 9:17-8 (1995); and Nadkarni et al., xe2x80x9cPreparation and biological activity of N-sulfonated chondroitin and dermatan sulfate derivatives,xe2x80x9d Carbohydrate Res., 290:87-96 (1996), the contents of which are hereby incorporated by reference.
D. Dermatan Sulfate Derivatives
Native dermatan sulfate (DS) is a better anticoagulant than heparin and is better able to facilitate inhibition of surface-bound thrombin. The specific heparin cofactor II (HCII)-mediated anti-thrombin (IIa) activity of DS has been significantly increased in one dermatan sulfate, Intimatan (CL-03135).
Smith degradation of Intimatan affords a fragment (Intimatan RD) with most of its HCII-mediated anti-IIa activity intact and with aldehyde terminal groups. RD reacts with primary amines to give labile Schiff-bases that can be converted into stable secondary amines by reduction with sodium cyanoborohydride. The anti-IIa activity of Intimatan is less than 60 u/mg, whereas the activity of RD is less than 40 u/mg.
E. Heparan Sulfates
Heparan sulfate, otherwise known as heparitin sulfate or heparin monosulfate, is a generic term describing polysaccharides which are linear and consist of N-acetylated [xe2x86x924) alpha- D-GlcNpS-(1xe2x86x924)-xcex2xcex2-D-GlcAp or alpha-L-ldoAp (1xe2x86x92] that are arranged mainly in a segregated manner. Approximately 25% of the total polymer is initially formed by alternating arrangements of the two disaccharide units, xe2x86x924)alpha-D-GlcNps(1xe2x88x92 greater than 4)UAp (1xe2x86x924) alpha-D-GlcNpAc(1xe2x86x924)UAp(1xe2x86x924)alpha-D-GlcAp(1xe2x86x92. The polymer is formed as a repeating xe2x86x924)alpha-D-GlcNpAc(1xe2x86x924)-xcex2xcex2-D-GlcAp (1xe2x86x92disaccharide sequence that is attached to a serine residue of a core protein through a tetrasaccharide, glucuronosyl xe2x86x92galactosyl xe2x86x92galactosyl xe2x86x92xylosyl, linkage region. It then undergoes partial N-deacetylation followed by N-sulfation of the newly exposed amino groups, partial C-5 epimerization of D-GlcAp to L-IdoAp and O-sulfation. O-sulfates are always found in proximity to N-sulfates which enhances the clustering of the sulfate residues and the heterogeneity in chemical composition and charge density of heparan sulfate. Suitable heparan sulfates are disclosed, for example, in Griffin et al., xe2x80x9cIsolation and characterization of heparan sulfate from crude porcine intestinal mucosa peptidoglycan,xe2x80x9d Carbohydrate Res., 276:183-197 (1995), the contents of which are hereby incorporated by reference.
F. Heparin Derivatives
Deaminative hydrolysis of unfractionated heparin with nitrous acid selectively cleaves the glycosidic bonds of the N-sulfated glucosamine residues with formation of di-, tetra-, hexa and higher saccharides terminated with 2,5-anhydro-D-mannose (AM) residues as reducing terminal groups. The terminal AM residues may be stabilized with sodium borohydride or coupled to an aminated surface by reductive amination.
Periodate causes the cleavage of carbon-carbon bonds if both adjacent carbons bear hydroxyl groups, or a hydroxyl group and an amino group. Unsulfated uronic acid residues in heparin are susceptible to periodate oxidation or Smith degradation. Fragments from periodate-oxidized heparin are larger than those obtained by nitrous acid degradation, reflecting relatively low contents of nonsulfated uronic acids. Those heparins containing aldehyde (CHO) moieties undergo reversible Schiff-base reactions with organic amines, and when treated with sodium cyanoborohydride, the Schiff base intermediate can be reduced to its corresponding amine forming an irreversible bond. In both these instances, the ATIII-binding site remains functionally intact.
Suitable heparin derivatives are described, for example, in Kosakai et al., xe2x80x9cIsolation and Characterization of Sulfated Disaccharides from the Deamination Products of Porcine Heparin,xe2x80x9d J. Biochem., 83:1567-75 (1978); Braswell, xe2x80x9cHeparin: Molecular Weight and Degradation Studies,xe2x80x9d Biochim. Biophys. Acta, 158:103-106 (1968); Fransson and Lewis, xe2x80x9cRelationship between anticoagulant activity of heparin and susceptibility to periodate oxidation,xe2x80x9d FEBS Lett., 97: 119-23 (1979); Nagasawa and Inoue, xe2x80x9cDe-N-sulfation,xe2x80x9d Meth. Carbohydrate Chem., VIII: 291-4 (1980); and Liu et al., xe2x80x9cNew Approaches for the Preparation of Hydrophobic Heparin Derivatives,xe2x80x9d J. Pharm. Sci. 83: 1034-1039 (1984), the contents of which are hereby incorporated by reference.
G. Heparin Fractions
Much of the heparin structure can be represented as a repeating trisulfated disaccharide. A pentasaccharide- containing trisulfated glucosamine residues represents the proposed structure of porcine intestinal mucosa heparin that specifically binds to antithrombin III. About a third of the molecules in unfractionated heparins contain this structure. The remaining 70% has no ATIII-dependent anti-clotting activity, but mediates the inhibition of thrombin through heparin cofactor II. The pentasaccharide sequence by itself is structurally incapable of inhibiting thrombin because molecules of at least 18 saccharides are required for simultaneous binding of heparin to ATIII and thrombin. As compared to unfractionated heparin, the fractions have either reduced or increased ATIII-mediated inhibition of thrombin and anti-Factor Xa activity.
Suitable heparin fractions are disclosed, for example, in Choay et al., xe2x80x9cStructural studies on a biologically-active hexasaccharide obtained from heparin,xe2x80x9d Ann. NY Acad. Sci., 370:644 (1981); and Laurent et al., xe2x80x9cThe molecular weight dependency of the anticoagulant activity of heparin,xe2x80x9d Biochem. J., 175:691(1978), the contents of which are hereby incorporated by reference.
H. Heparin Fragments
Heparin fragments are the result of enzymatic or chemical cleavage in which (I) heparinase cleaves unfractionated heparin linkages between N-sulfated glucosamine and uronic acid with the formation of oligosaccharides bearing 4,5-unsaturated uronic acid at the non-reducing end; (ii) esters of the iduronic carboxyl groups of heparin are subjected to xcex2xcex2-elimination at alkaline pH with the formation of 4,5-unsaturated uronic acid at the non-reducing end; (iii) nonsulfated uronic acid residues of heparins are cleaved by oxidation with either nitrous acid or periodate, followed by reduction of the resulting aldehyde(s) with borohydride and hydrolysis under mild acidic conditions, thus producing end groups with the remnant of the nonsulfated uronic acid; (iv) the glycosidic bonds of heparin are cleaved by a radical mechanism using hydrogen peroxide, known as oxidative-reductive depolymerization, resulting in fragments having reducing end groups, and (v) heparin chains are cleaved concomitant with sulfation by the action of a mixture of sulfuric and clorosulfonic acids.
Low-molecular weight heparins (LMWHs) are fragments of conventional porcine-derived heparin. LMWHs were developed to provide more selective inhibition of enzyme function and reduce adverse effects. Heparin fragmentation produces products which maintain activity against factor Xa and release antithrombotic factors, but have significantly less activity against factor IIa. As a result, treatment with LMWHs provides antithrombotic effects with less anticoagulant effect, lessening the risk of hemorrhage.
Relative to unfractionated heparin, LMWHs exhibit a reduced ability to prolong thrombin inhibition and an enhanced capacity to inhibit factor Xa, thereby contributing to an improved anti-thrombotic effect. The minimum size for anti-thrombin III (ATIII) binding is a pentasaccharide. However, the pentasaccharide-ATIII complex only inhibits factor Xa and not thrombin as heparin oligosaccharides of  less than 5400 D are without cofactor activity for thrombin. Studies have shown that when comparing the rate of thrombosis development or complications, LMWHs have demonstrated similar efficacy as heparin.
One advantage of using LMWHs is that there is a reduced incidence of hemorrhage and HIT relative to heparin.
There are several LMWH products currently on sale in the United States or being actively investigated. These include Enoxaparin(trademark) (Rhone-Poulenc Rorer), Dalteparin(trademark) (PharmaciaandUpjohn), Ardeparin(trademark) (Wyeth-Ayerst) and Centaxarin(copyright) FH. Centaxarin(copyright) FH (Ardeparin sodium, ML-009723) is the sodium salt of LMWH obtained by the oxidative-reductive depolymerization of porcine mucosal heparinic acid pursuant to FDA Drug Master File 7952.
Enoxaparin(trademark) is typically administered by subcutaneous injection. The recommended adult dose is 30 to 40 mg given twice daily. Dalteparin(trademark) has a longer elimination half-life than Enoxaparin(trademark), allowing once daily dosing. Like Enoxaparin(trademark), Dalteparin(trademark) is administered subcutaneously. The dose is based on units of anti-Xa activity. The recommended adult dose for Dalteparin(trademark) is 2,500 to 5,000 anti-factor Xa units given once daily. Ardeparin(trademark) is dosed based on patient weight. The recommended adult dose is 50 anti-Xa units/kg administered every 12 hours. Disaccharide analysis qualifies FH as a LMWH with substantial retention of the xe2x80x9cinternalxe2x80x9d heparin structure and without any xe2x80x9cmodifiedxe2x80x9d residues.
Other suitable heparin fragments include those disclosed in Fareed et al., xe2x80x9cAT-III Dependence on the biochemical and pharmacologic actions of a low molecular weight heparin,xe2x80x9d Thromb. Haemostas., 69:1269 (1993); Schxc3xa4xc3xa4fer et al., xe2x80x9cAnticoagulant and lipasemic profile of a new low molecular weight heparin fragment in man,xe2x80x9d Thromb. Haemostas. 69:2402 (1991); and Malinowski et al., xe2x80x9cComparative pharmacologic studies on a new low molecular weight heparin (ML-009723) and Enoxaparin,xe2x80x9d Thromb. Haemostas.69:1260 (1993), the contents of which are hereby incorporated by reference.
I. Heparin Salts
Heparin salts, usually from porcine intestinal mucosa, are polydisperse in chain length and heterogeneous in degree and type of sulfation. Heparin salts are strongly anionic polyelectrolytes and are effective in functions involving binding and release of micro-ions.
Heparin can form salts with both monovalent cations, such as sodium, and divalent cations, such as calcium. Divalent cations such as calcium bind more strongly to heparin than monovalent counterions.
J. Mixtures of Heparin-Like Substances
Mixtures of heparin-like substances can be used. One example of such a mixture is Danaparoid(trademark) sodium. Danaparoid sodium is an alternative anticoagulant in patients who develop heparin-induced thrombocytopenia (HIT) from heparin therapy. Danaparoid is a low molecular weight heparinoid derived from porcine gut mucosa. Its active components consist of heparan sulfate, dermatan sulfate and chondroitin sulfate. The major difference between Danaparoid and other low molecular weight heparins (LMWH) is that Danaparoid is devoid of heparin or heparin fragments. However, similar to LMWHs, it exerts its antithrombotic effect principally through anti-thrombin III-mediated inhibition of factor Xa and, to a much lesser extent, thrombin. The cross-reactivity of Danaparoid with heparin-induced antibodies is reportedly less than 10%.
K. Targeted Heparin and Heparin-Like Substances
Heparin and heparin-like substances can be targeted to the human thrombus with antibodies, such as the high affinity fibrin antibody DD-3B6/22. Binding multifinctional targeted anticoagulants to the thrombus allows the inhibition of other components of thrombus associated procoagulant activity such as the Factor Xa dependent generation of thrombin and the inhibition of platelet activation. Various monoclonal antibodies (such as DD-3B6/22) have been developed which bind to crosslinked fibrin found in clots in situ, often with relatively high affinity (on the order of 10xe2x88x929M or less). (See, for example, J. Nuc. Med. 35:195-202 (1994), the contents of which are hereby incorporated by reference.)
L. Other Anti-Coagulants
The anti-coagulants discussed below are not intended to be construed as heparin-like substances, and are not equivalents for heparin or heparin-like substances for purposes of the present invention. However, these can optionally be included in the compositions and used in the methods disclosed herein.
Ancrod is an anticoagulant derived from snake venom. Ancrod does not cross-react with heparin-induced antibodies. However, patients can develop neutralizing anti-ancrod antibodies over time. Ancrod reduces fibrinogen levels, thereby decreasing plasma viscosity. It does not inhibit thrombin, which may limit its use in some HIT patients, particularly those who have disseminated intravascular coagulation (DIC) or septicemia.
Warfarin is another widely used anticoagulant. Warfarin has a relatively slow onset of action, taking up to 5 days for full anticoagulant effect.
Hirudin is the active anticoagulant in the saliva of leeches. Hirudin and its peptide analogues, hirulog and argatroban, are also commonly used as anticoagulants.
Other widely used thrombolytic agents or platelet inhibiting substances include streptokinase, urokinase, tissue plasminogen activator, acetyl salicylic acid, coumadin, coumarin, and dipyridamole.
The above anti-coagulants can also be targeted as discussed above with respect to heparin and heparin-like substances.
Three major classes of adenosine receptors, classified as A1, A2, and A3, have been characterized pharmacologically. A1 receptors are coupled to the inhibition of adenylate cyclase through Gi proteins and have also been shown to couple to other second messenger systems, including inhibition or stimulation of phosphoinositol turnover and activation of ion channels. A2 receptors are further divided into two subtypes, A2A and A2B, at which adenosine agonists activate adenylate cyclase with high and low affinity, respectively. The A3 receptor sequence corresponds to a novel, functional adenosine receptor.
Adenosine binds to all four adenosine receptor sites in a non-specific manner. Adenosine has a relatively short half-life in vivo (less than about 30 seconds), although it is effective at relatively low doses. In one embodiment, adenosine solutions are administered intravenously over an extended period of time to produce the desired effect. Chronic administration of adenosine over a period of a week or more has an angiogenic effect, which is increased by the co-administration of heparin or heparin-like substances.
Agonism at the A2a and A2b receptors is responsible for the angiogenic effect. Adenosine receptor agonists have been developed which have high affinity and selectivity for these receptors. Suitable A2 agonists include 2-[p-(2-carboxyethyl)phenethyl-amino]-5xe2x80x2-N-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2-receptor agonist, 4-[2-[[6-Amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid, a selective adenosine A2 receptor agonist, and CV-1808 (Glaxo Wellcome). Other A2 agonists include those described in Niiya et al., J. Med. Chem., 35:4557-4561(1992); Ueeda et al., J. Med. Chem., 34:1340-1344 (1991), Niiya et al., J. Med. Chem., 35:4562-4566 (1992), and Ueeda et al., J. Med. Chem., 34(4):1334-1339 (1991), the contents of which are hereby incorporated by reference.
The use of adenosine A1 and A3 receptor agonists is associated with cardioprotection. Accordingly, the compositions can optionally include A1 and A3 receptor agonists in addition to the adenosine A2 receptor agonists. Suitable A1 agonists include N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, 2-chloroadenosine, CPA, R-PIA,GR 79236 (Glaxo Wellcome). Suitable A3 agonists include IB-MECA (1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-xcex2-D-ribofuranuronamide), a selective A3 adenosine receptor agonist, R-PIA ((R)-N6-(phenylisopropyl)adenosine), and NECA (5xe2x80x2-N-ethylcarboxamido adenosine) (Glaxo Wellcome).
Other adenosine receptor agonists include those taught in U.S. Pat. Nos. 3,819,612, 3,819,613, 4,954,504, 5,034,381, 5,063,233, 5,140,015 , 5,278,150, and 5,593,875, the contents of each of which are incorporated herein by reference.
Methods of Treatment
The adenosine A2 receptor agonist and heparin and/or heparin-like substance can be used in a method for promoting angiogenesis in a patient in need thereof. The method involves the co-administration of an effective amount of heparin or a heparin-like substance and an effective amount of an adenosine A2 receptor agonist in low, daily dosages for a week or more. The method may be used as a treatment to restore cardiac function after a myocardial infarction. The method may also be used to improve blood flow in patients with coronary artery disease suffering from myocardial ischemia or inadequate blood flow to areas other than the heart, for example, peripheral vascular disease, for example, peripheral arterial occlusive disease, where decreased blood flow is a problem.
The compounds can be administered via any medically acceptable means which is suitable for the compound to be administered, including oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration. For example, adenosine has a very short half-life. For this reason, it is preferably administered intravenously. However, adenosine A2 agonists have been developed which have much longer half-lives, and which can be administered through other means. Heparin and heparin-like substances can be administered, for example, intravenously or by subcutaneous administration.
In some embodiments, the adenosine A2 receptor agonist and heparin or heparin-like substance are administered via different means of administration. For example, the heparin or heparin-like substance can be administered in a once-daily subcutaneous injection, and the adenosine A2 receptor agonist can be administered intravenously for a given period of time.
The amounts of the adenosine A2 receptor agonist and heparin or heparin-like substance required to be effective in stimulating angiogenesis will, of course, vary with the individual being treated and is ultimately at the discretion of the physician. The factors to be considered include the condition of the patient being treated, the efficacy of the particular adenosine A2 receptor agonist being used, the nature of the formulation, and the patient""s body weight. Occlusion-treating dosages of heparin or a heparin-like substance and an adenosine A2 receptor agonist are any dosages that provide the desired effect. However, a suitable occlusion-treating dose of heparin (or heparin-like substance) and an adenosine A2 receptor agonist is in the range of about 5000 to about 10,000 U/d heparin and about 40 mg to about 80 mg of an adenosine A2 receptor agonist for ten days. While it possible to administer heparin (or a heparin-like substance) and an adenosine A2 receptor agonist simultaneously, preferably heparin (or a heparin-like substance) is given as a bolus about twenty minutes before starting the administration of the adenosine A2 receptor agonist.
Typically, when heparin is used, it is infused as a bolus of about 15,000 U about 15 minutes prior to the adenosine A2 receptor agonist administration. When adenosine is used as the adenosine A2 receptor agonist, it is then infused for about 5 to about 8 minutes at a rate of about 140 xcexcg/Kg/min (based on body weight). Thus, a total dose for a 80 Kg patient is about 67 mg. This dosage regiment is repeated daily for about 10 days. The adenosine A2 receptor agonist-heparin infusions can be used to stimulate angiogenesis in patients with symptomatic coronary artery disease in place of other more invasive and expensive therapies such as angioplasty or even coronary artery bypass grafting surgery (CABG).
Effective doses for heparin-like substances and for adenosine A2 receptor agonists other than adenosine are well known to those of skill in the art, and, in some cases, have been described above. Generally, for heparin-like substances, an effective dose is that which maintains the anti-Xa level between 0.5 and 1.0 units/ml. This range has been shown to optimize antithrombotic activity while avoiding adverse effects. Suitable effective dose for adenosine A2 receptor agonists other than adenosine are typically in the range of about 0.1 xcexcg/kg to about 10 mg/kg body weight per day, preferably in the range of about 1 mg/kg to about 3 mg/kg per day.
The adenosine A2 receptor agonist can be administered to a patient in any pharmacologically and pharmaceutically acceptable form. Preferably, the agonist is administered via a continuous, intravenous infusion, more preferably, in an isotonic, aqueous solution. Both the heparin (and/or the heparin-like substance) and the adenosine A2 receptor agonist can be administered in sterile, buffered, dilute aqueous solutions. Preferably, excipients such as preservatives, stabilizers, and antioxidants are added to these solutions. The prototypical adenosine A2 receptor agonist, adenosine, per se, can be obtained from several sources, e.g., from Fujisawa under the trademark Adenoscan(copyright). Likewise, pharmaceutical forms of heparin and heparin-like substances, such as sodium heparin, are also readily available.
The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary. For example, for a 75 kg mammal, a dose range for the adenosine A2 receptor agonist would be about 75 mg to about 220 mg per day, and a typical dose would be about 150 mg per day. If discrete multiple doses are indicated, treatment might typically be 50 mg of a compound given 3 times per day. In one embodiment, the adenosine A2 agonist alone causes the beneficial effect, without the need for co-administration of heparin or a heparin-like substance.
Formulations
The compounds described above are preferably administered in a formulation including an adenosine A2 receptor agonist and heparin and/or a heparin-like substance together with an acceptable carrier for the mode of administration. Any formulation or drug delivery system containing the active ingredients, which is suitable for the intended use, as are generally known to those of skill in the art, can be used. Suitable pharmaceutically acceptable carriers for oral, rectal, topical or parenteral (including subcutaneous, intraperitoneal, intramuscular and intravenous) administration are known to those of skill in the art. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Formulations suitable for parenteral administration conveniently include sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, 5% dextrose in distilled water or saline. Useful formulations also include concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration above.
For enteral administration, a compound can be incorporated into an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients. Such accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.
Alternatively, the compound may be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. U.S. Pat. No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, xe2x80x9cLiposomes,xe2x80x9d Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).
Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents of which are hereby incorporated by reference.
In one embodiment, the heparin or heparin-like substance and/or the adenosine A2 agonist can be formulated into a liposome or microparticle which is suitably sized to lodge in capillary beds following intravenous administration. When the liposome or microparticle is lodged in the capillary beds surrounding ischemic tissue, the agents can be administered locally to the site at which they can be most effective. Suitable liposomes for targeting ischemic tissue are generally less than about 200 nanometers and are also typically unilamellar vesicles, as disclosed, for example, in U.S. Pat. No. 5,593,688 to Baldeschweiler, entitled xe2x80x9cLiposomal targeting of ischemic tissue,xe2x80x9d the contents of which are hereby incorporated by reference.
Preferred microparticles are those prepared from biodegradable polymers, such as polyglycolide, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.
In one embodiment, the formulations are administered via catheter directly to the inside of blood vessels. The administration can occur, for example, through holes in the catheter. In those embodiments wherein the active compounds have a relatively long half life (on the order of 1 day to a week or more), the formulations can be included in biodegradable polymeric hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016 to Hubbell et al. These polymeric hydrogels can be delivered to the inside of a tissue lumen and the active compounds released over time as the polymer degrades. If desirable, the polymeric hydrogels can have microparticles or liposomes which include the active compound dispersed therein, providing another mechanism for the controlled release of the active compounds.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.
The formulations can optionally include additional components, such as various biologically active substances such as growth factors (including TGF-xcex2, basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), transforming growth factors xcex1 and xcex2 (TGF xcex1 and xcex2), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF)), antivirals, antibacterials, antiinflammatories, immunosuppressants, analgesics, vascularizing agents, cell adhesion molecules (CAM""s), and anticoagulants other than heparin or heparin-like substances.
In addition to the aforementioned ingredients, the formulations may further include one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
Determination of the Degree of Activity for the Compounds
The activity of the compounds can be readily determined using no more than routine experimentation using any of the following assays.
A. Heparin or Heparin-Like Substances:
The HCII-mediated anti-IIa activity of a heparin-like substance can be determined in a purified system by incubating a sample solution with purified HCII and thrombin. Chromogenic substrate can be added and the amidolytic thrombin activity measured at 405 nm.
B. Adenosine Receptor Agonists
The activity and selectivity of the adenosine receptor agonists for each of the adenosine receptors can be readily determined using no more than routine experimentation using any of the following assays.
Binding Assays.
The prototypical allosteric enhancer PD 81,723, (see Bruns et al., Mole. Pharm., 38:939 (1990), Cao et al., Gen Pharmac. 26:1545 (1995), and Amoah-Apraku et al., J Pharm. Exper. Ther. 266(2):611(1993)) has both enhancing and inhibitory activity at the A1AdoR. Therefore, the effect of adenosine agonists can be determined on both the agonist [3H]CCPA and the antagonist [3H]CPX binding to membranes prepared from CHO cells stably expressing the human A1 AdoR (CHO-huA1 AdoR). The enhancing activity can be estimated by the magnitude of the increase in [3H]CCPA binding whereas the inhibitory and (or antagonistic) activity can be evaluated by the potency of the agonists to compete for the specific binding of [3H]CPX. A suitable method for preparing membranes of CHO cells expressing huA1 AdoR, and the protocols for the radioligand binding assays is described by Kollias-Baker et al., (JPET, 281, 761(1997) and Circ. Res., 75, 961 (1994)).
Similar assays for assaying A2 and A3 activity are well known to those of skill in the art.